Safety and effectiveness of RBD-specific polyclonal equine F(ab´)2 fragments for the treatment of hospitalized patients with severe Covid-19 disease: A retrospective cohort study

Background Passive immunotherapy has been evaluated as a therapeutic alternative for patients with COVID-19 disease. Equine polyclonal immunotherapy for COVID-19 (EPIC) showed adequate safety and potential efficacy in a clinical trial setting and obtained emergency use authorization in Argentina. We studied its utility in a real world setting with a larger population. Methods We conducted a retrospective cohort study at “Hospital de Campaña Escuela-Hogar" (HCEH) in Corrientes, Argentina, to assess safety and effectiveness of EPIC in hospitalized adults with severe COVID-19 pneumonia. Primary endpoints were 28-days all-cause mortality and safety. Mortality and improvement in modified WHO clinical scale at 14 and 21 days were secondary endpoints. Potential confounder adjustment was made by logistic regression weighted by the inverse of the probability of receiving the treatment (IPTW) and doubly robust approach. Findings Subsequent clinical records of 446 non-exposed (Controls) and 395 exposed (EPIC) patients admitted between November 2020 and April 2021 were analyzed. Median age was 58 years and 56.8% were males. Mortality at 28 days was 15.7% (EPIC) vs. 21.5% (Control). After IPTW adjustment the OR was 0.66 (95% CI: 0.46–0.96) P = 0.03. The effect was more evident in the subgroup who received two EPIC doses (complete treatment, n = 379), OR 0.58 (95% CI 0.39 to 0.85) P = 0.005. Overall and serious adverse events were not significantly different between groups. Conclusions In this retrospective cohort study, EPIC showed adequate safety and effectiveness in the treatment of hospitalized patients with severe SARS-CoV-2 disease.


Data collection process for EPIC cohort group
All data from EPIC patients in the cohort were retrieved from the prospective, structured registry 23 designed within the frame of ANMAT ś Provision 4622/12 regarding authorization under special 24 conditions. In addition, a review of the HCEH ś electronic medical records was performed for all 25 selected patients in order to complete other data of interest. For the selection of the exposed patients 26 a complete list of admissions included in the registry from the HCEH' between January 27th and 27 April 17th, 2021, was reviewed. Patients with severe disease (defined as having respiratory rate of 28 more than 30/min, or oxygen saturation <94% on room air at sea level, or a ratio of arterial partial 29 pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) <300 mm Hg, or lung compromise 30 of more than 50%) at the initiation of EPIC treatment within 24 hours of hospitalization were 31 identified. Patients having moderate disease (including patients that received supplementary 32 oxygen within 24 hours of hospitalization but with no documentation of tachypnea, oxygen 33 desaturation or lung compromise above 50% through imaging) were excluded from this cohort 34 group, as well as patients with mild and critical disease (including patients admitted to ICU, 35 receiving mechanical ventilation or requiring inotropic drugs since hospital admission). 36 Review of the electronic medical records was started with the patients with earlier admission 37 (January 27th, 2021) and moved forward toward the more recent admission dates until reaching 38 the target sample of patients that had received at least one dose of EPIC. The medical data 39 collection team retrieved the additional data of interest in a specific structured form developed for 40 that purpose. After verification of completeness, a different team added the information to the 41 electronic study database in an anonymized fashion. Data collection process for the "Control" cohort group 44 For the selection of the "Control" patient group a complete list of all patients within 18and 79-45 year-old admitted to the "HCEH" between November 25th, 2020 and January 21st, 2021 was 46 obtained.

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The medical data collection team thoroughly reviewed each electronic medical record in order to 48 evaluate the selection criteria and completed the specific structured forms with the clinical data of 49 the selected patients. This review was performed starting with the more recent admissions and 50 moved backward until reaching the target sample complying the selection criteria. Again, a 51 verification of completeness was performed before a different data team added this information to 52 the electronic study database in an anonymized fashion. electronic case report forms that remained inalterable and protected from that moment onwards.

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Finally, a complete analysis of the data was carried out following the Statistical Analysis Plan.

Statistical Analysis Plan
Operationalization of variables variables, while presence and type of comorbidities, prior use of convalescent plasma, diagnostic 66 method, respiratory rate (≤20 or >20/min), requirement of supplementary oxygen and oxygen   All patient characteristics from the EPIC and Control groups were compared in order to detect 77 potential confounders. Categorical variables were compared using the Chi square or Fisher's exact 78 test and quantitative characteristics were compared using Student's T test or Mann Whitney's U 79 test according with assumptions.

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Although the study design itself may equalize overall EPIC and Control patient characteristics, 81 since the authorization date for the use of EPIC at the HCEH is independent from the patient's 82 characteristics, all association measurements were presented either as raw data and adjusted for 83 inverse probability of treatment weighting (IPTW) and potential confounders, defining a doubly robust method for estimation of the potential causal effect of the intervention of interest in 85 comparison with the "non-exposed" cohort patients.

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Given the observational nature of the study, all patient characteristics showing statistically 87 significant differences were considered as potential confounders in addition to all variables 88 identified "a priori" by researcher´s medical criteria. Taking into consideration the mortality events as right-censored events, there were no competing 92 events for the primary outcome and its cumulative incidence was estimated using the Kaplan Meier 93 method. Cumulative incidence curves according to time were presented at 28 days of follow up.

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Mortality cumulative incidence at days 14, 21 and 28 were estimated with 95% confidence 95 intervals (CI95%). Median time to event and 25-75% percentiles were calculated. A comparison 96 of survival curves between the two cohort groups was made with Cox-Mantel hypothesis test 97 considering null hypothesis as survival curve overlap between EPIC and Control.

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An univariate Cox proportional hazard regression model was used for estimation of the Hazard 99 Ratio (HR) between cohort groups using death as result variable. In addition, an adjusted HR was 100 obtained through the same regression model using IPTW and weighting by potential confounders 101 (doubly robust approach). Both raw and adjusted HR were calculated with their respective CI95%.

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Both raw OR as well as weighted by IPTW and potential confounders were presented with their 113 respective CI95%.  Considering as null hypothesis an equal proportion of each secondary outcome between EPIC and 121 "Control" groups, a Chi square or Fisher's exact test were used according to assumptions. Bivariate 122 logistic regression model was used to estimate the raw OR of both cohort groups. Adjusted HR 123 were obtained through the same regression model using IPTW and weighting by potential confounders (doubly robust approach). Both raw and adjusted HR were calculated with their 125 respective CI95%. A multivariate logistic regression model was used for calculation of the propensity score using 145 exposition to EPIC as dichotomous response variable. All other identified potential causes of 146 exposition or death were included as explanatory variables. In addition, all unbalanced variables 147 and those considered as potential predictors of EPIC use or reflecting changes in diagnosis, staging, 148 concomitant treatment or support measures between cohort groups were included as explanatory 149 variables. 150 We estimated the propensity score (PS) of EPIC exposure using a logistic regression model with 151 EPIC exposure as dependent variable and the following potential predictors of treatment: gender 152 at birth, age, clinical parameters at cohort admission (respiratory rate, heart rate, body temperature, interest, the following subgroups were pre-specified: gender at birth, age category groups (less 178 than 65, or between 65 and 79 years old), time from symptoms initiation (less or more than 3 days, 179 less or more than 5 days or between 5 and 10 days), obesity, presence and number of main 180 comorbidities (immunosuppression, diabetes, arterial hypertension, cardiovascular disease) and 181 obesity.

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All tests were two-sided, and a P value < 0.05 was considered statistically significant. All statistical

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Population characteristics 189 A complete description of patients´ comorbidities at cohort entry is shown in S1 Table. 190 191

Secondary analyses 192
Patient's discharge at day 14 was significantly greater in the EPIC group than in the Control group 193 -280 (79,9%) vs 281 (63%) OR 1.46 (95% CI 1.07 to 1.98), P=0.016 for IPTW adjustment. There 194 were no differences between cohort groups in the rest of the secondary outcomes evaluated.

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Other than mortality and WHO-modified ordinal clinical scale results, the complete results of 196 secondary outcomes are provided in S2 Table. 197 198 Sensitivity analyses 199 Sensitivity analysis for mortality performed to subjects that received two complete doses of EPIC  Table. 207 We performed an additional sensitivity analysis with the respiratory rate and oxygen saturation 208 variables considered as continuous. In such case the OR for the primary outcome was 0.75 (95% 209 CI 0.51 to 1.10) P=0.142 and the HR 0.78 (95% CI 0.55 to 1.10) P=0.160 for the IPTW adjustment,